We plan to develop the novel vaccines targeting the released proteins/peptides (secretome) during the early infection of Bacillus anthracis. In the past years, we had established the proteomic techniques to identify novel anthrax antigens. Several vector-based vaccines encoding these novel anthrax antigens have been constructed in our laboratory. In addition, we have published a proteome of Bacillus anthracis by identifying spore proteins associated with germination and early outgrowth. However, the released proteins/peptides (secretome) of Bacillus anthracis are not included in our and other published anthrax proteomes. In this proposal, we will mainly employ the mass spectrometric techniques to identify the anthrax secretomes with/without protein separation by 2-D electrophoresis. We will collect the secretomes from anthrax-infected mice using capillary ultrafiltration (CDF) probes in order to capture the in vivo low abundant and secretory proteins/peptides. The CDF probes are a novel technique recently developed in our laboratory to collect low abundant and secretory proteins from mice. We plan to emphasize on the proteins/peptides released during the early stages of anthrax infection. Our hypothesis is that vaccines targeting the early stages of anthrax infection (upstream events) will block the downstream events including the production of protective antigen (PA), lethal factor (LF), and edema factor (EF). In order to construct more effective vaccines, we will determine the cytotoxicities and immunogenicities of anthrax secretomes. In parallel, we will evaluate the efficacies of vaccines which encoded secretomes of early anthrax infection as compared to current PA/LF-based vaccines. Lastly, we have identified a novel toxin called camelysin-like protein (CLP) which exerts immunogenic and is released from dormant spores during the early stage of the anthrax life cycle. This protein will thus serve as a positive control to evaluate novel antigens identified in the proposed studies.